*** Primer 7 is VF2 in BioBricks. Primer 60 is its reverse compliment. In a biobrick vector, it appears light gray for 7 and dark gray for 60. pCM66 happens to have this same sequence in the region upstream from the multiple cloning site, except it is REVERSED. Both primers will appear red as they bind in the opposite direction expected.

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** Primer 7 is VF2 in BioBricks. Primer 60 is its reverse compliment. In a biobrick vector, it appears light gray for 7 and dark gray for 60. pCM66 happens to have this same sequence in the region upstream from the multiple cloning site, except it is REVERSED. Both primers will appear red as they bind in the opposite direction expected.

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*** I designed some primers for a Kan casette. The Kan casette in pCM66 is read in the reverse direction, so all the primers built for a forward Kan casette appear red. [[image:2013_05_08_Kan_casette.jpg||thumb|center|Kan primers binding in the reverse direction intended appear red]]

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** I designed some primers for a Kan casette. The Kan casette in pCM66 is read in the reverse direction, so all the primers built for a forward Kan casette appear red. [[image:2013_05_08_Kan_casette.jpg||thumb|center|Kan primers binding in the reverse direction intended appear red]]

== Skills I'm developing ==

== Skills I'm developing ==

Revision as of 09:40, 8 May 2013

Janet B. Matsen

Department of Chemical Engineering
Seattle, Washington

jmatsen@uw.edu

I am a 3rd year Chemical Engineering PhD student at the University of Washington working at Mary Lidstrom's Lab and it is so fun!

My first year was spent investigating methanotrophic metabolism in pure cultures and a model ecosystem in a team that combined transcriptomics, metabolomics, and single-cell observation. Currently I am helping engineer E. coli to make biofuel precursors from electricity and CO2.

I started the Lidstrom Lab OWW wiki and am addicted to posting what I learn! Amanda Smith is significant contributor as well. It has been a lot of fun for me to record what I have learned about lab techniques. It is also a fun place me to share tips/tricks and experiments that probe how important variables in a protocol are.

Tools to Share

This is an R script that converts the info in my list of primers into a file that I can use to annotate DNA files in APE with. It:

trims out sequences not intended for sequencing such as Gibson assembly primers

makes a label that combines the unique primer number, the melting temperature, and the letter F or R for forward or reverse, and an asterisk if you should consult the primer spreadsheet comments before using it

assigns colors in APE that communicate whether it primers in the forward direction or the reverse direction.

saves the info in the format APE needs, with the date it was generated in the title.

This allows me to instantly see where all of the primers I own bind to a DNA sequence for a given project I am working on. It also allows me to share these primers very easily; by sharing the file it outputs allows my lab mates to instantly see if I have any primers that can be used in their project. It has been very handy for them!

I am happy to help friends modify this script to be useful with their own primer libraries! No R experience is necessary.

Anyone can access my most current primer "Annotation Feature Library" here. You can also see the files used to generate it there.

Use notes

If the primer binds in the forward direction, the primer will be light gray

If the primer binds in the reverse direction, it will be dark gray

If the primer binds in the reverse direction that it was designed for, it will appear red.

demo of APE primer library tool

Examples:

Primer 7 is VF2 in BioBricks. Primer 60 is its reverse compliment. In a biobrick vector, it appears light gray for 7 and dark gray for 60. pCM66 happens to have this same sequence in the region upstream from the multiple cloning site, except it is REVERSED. Both primers will appear red as they bind in the opposite direction expected.

I designed some primers for a Kan casette. The Kan casette in pCM66 is read in the reverse direction, so all the primers built for a forward Kan casette appear red.